Striking advances in the areas of bioinformatics, mass spectrometry and parallel yeast two-hybrid analyses have made it possible to deduce the protein components of almost any multi-protein complex. However, there remains no reliable method by which the organization of the proteins in the complex can be deduced, a central issue in eventually understanding the mechanism of action of any given complex. A related problem is to understand how proteins that regulate a complex through direct physical interactions dock with the protein machine. The major goal of this project is to develop a general protocol to address these problems. This will be done by elaborating on a novel protein cross-linking chemistry developed in my laboratory over the last two years. In this chemistry, water-soluble metal complexes are activated by brief photolysis with visible light in the presence of an electron acceptor. This results in the photo-oxidation of the complex, which in turn is thought to oxidize accessible tyrosine or tryptophan residues in proteins, creating radical intermediates. These radicals can couple to nearby side chains from partner proteins, probably by a variety of mechanisms, thus leading to covalent protein cross-linking. The reaction is fast, efficient and cross-links only proteins that are closely associated. We propose to employ this chemistry in combination with two-dimensional gel electrophoresis/mass spectrometry techniques to elucidate the architecture of the yeast 26S proteasome. In parallel, the cross-linking protocol (and related oxidative labeling reactions) will be employed to identify the specific sub-units contacted by a variety of exogenous ligands that are known to bind to the proteasome. Finally, a new aspect application of this oxidative chemistry is proposed: the development of a general method by which to identify the protein target(s) of bioactive small molecules.